Assays for protein kinases using fluorescent protein substrates

ABSTRACT

This invention provides assays for protein kinase activity using fluorescent proteins engineered to include sequences that can be phosphorylated by protein kinases. The proteins exhibit different fluorescent properties in the non-phosphorylated and phosphorylated states.

BACKGROUND OF THE INVENTION

[0001] This invention relates to the field of enzymatic assays and, inparticular, assays for protein kinase activity involving modifiedfluorescent proteins.

[0002] Protein phosphorylation is one of the most important generalmechanisms of cellular regulation. Protein phosphorylation commonlyoccurs on three major amino acids, tyrosine, serine or threonine, andchanges in the phosphorylation state of these amino acids withinproteins can regulate many aspects of cellular metabolism, regulation,growth and differentiation. Changes in the phosphorylation state ofproteins, mediated through phosphorylation by kinases, ordephosphorylation by phosphatases, is a common mechanism through whichcell surface signaling pathways transmit and integrate information intothe nucleus. Given their key role in cellular regulation, it is notsurprising that defects in protein kinases and phosphatases have beenimplicated in many disease states and conditions. For example, theover-expression of cellular tyrosine kinases such as the EGF or PDGFreceptors, or the mutation of tyrosine kinases to produce constitutivelyactive forms (oncogenes) occurs in many cancer cells. Drucker et al.(1996) Nature Medicine 2: 561-56. Protein tyrosine kinases are alsoimplicated in inflammatory signals. Defective Thr/Ser kinase genes havebeen demonstrated to be implicated in several diseases such as myotonicdystrophy as well as cancer, and Alzheimer's disease (Sanpei et al.(1995) Biochem. Biophys. Res. Commun. 212: 341-6; Sperber et al (1995)Neurosci. Lett. 197: 149-153; Grammas et al (1995) Neurobiology of Aging16: 563-569; Govoni et al. (1996) Ann. N.Y. Acad. Sci. 777: 332-337).

[0003] The involvement of protein kinases and phosphatases in diseasestates makes them attractive targets for the therapeutic intervention ofdrugs, and in fact many clinically useful drugs act on protein kinasesor phosphatases. Examples include cyclosporin A which is a potentimmunosuppressant that binds to cyclophilin. This complex binds to theCa/calmodulin-dependent protein phosphatase type 2B (calcineurin)inhibiting its activity, and hence the activation of T-cells. (Sigal andDumont (1992), Schreiber and Crabtree (1992)). Inhibitors of proteinkinase C are in clinical trails as therapeutic agents for the treatmentof cancer. (Clin. Cancer Res. (1995) 1:113-122) as are inhibitors ofcyclin dependent kinase. (J. Mol. Med. (1995) 73:(10):509-14.)

[0004] The number of known kinases and phosphatases are growing rapidlyas the influence of genomic programs to identify the molecular basis fordiseases have increased in size and scope. These studies are likely toimplicate many more kinase and phosphatase genes in the development andpropagation of diseases in the future, thereby making them attractivetargets for drug discovery. However current methods of measuring proteinphosphorylation have many disadvantages which prevents or limits theability to rapidly screen using miniaturized automated formats of manythousands of compounds. This is because current methods rely on theincorporation and measurement of ³²P into the protein substrates ofinterest. In whole cells this necessitates the use of high levels ofradioactivity to efficiently label the cellular ATP pool and to ensurethat the target protein is efficiently labeled with radioactivity. Afterincubation with test drugs, the cells must be lysed and the protein ofinterest purified to determine its relative degree of phosphorylation.This method requires high numbers of cells, long preincubation times,careful manipulation and washing steps (to avoid artifactualphosphorylation or dephosphorylation), as well as a method ofpurification of the target protein. Furthermore, final radioactiveincorporation into target proteins is usually very low, giving the assaypoor sensitivity. Alternative assay methods, for example based onphosphorylation-specific antibodies using ELISA-type approaches, involvethe difficulty of producing antibodies that distinguish betweenphosphorylated and non-phosphorylated proteins, and the requirement forcell lysis, multiple incubation and washing stages which are timeconsuming, complex to automate and potentially susceptible to artifacts.

[0005] Kinase assays based on purified enzymes require large amounts ofpurified kinases, high levels of radioactivity, and methods ofpurification of the substrate protein away from incorporated³²P-labelled ATP. They also suffer from the disadvantage of lacking thephysiological context of the cell, preventing a direct assessment of adrugs toxicity and ability to cross the cells plasma membrane.

[0006] Fluorescent molecules are attractive as reporter molecules inmany assay systems because of their high sensitivity and ease ofquantification. Recently, fluorescent proteins have been the focus ofmuch attention because they can be produced in vivo by biologicalsystems, and can be used to trace intracellular events without the needto be introduced into the cell through microinjection orpermeabilization. The green fluorescent protein of Aequorea victoria isparticularly interesting as a fluorescent indicator protein. A cDNA forthe protein has been cloned. (D. C. Prasher et al., “Primary structureof the Aequorea victoria green-fluorescent protein,” Gene (1992)111:229-33.) Not only can the primary amino acid sequence of the proteinbe expressed from the cDNA, but the expressed protein can fluoresce.This indicates that the protein can undergo the cyclization andoxidation believed to be necessary for fluorescence. The fluorescence ofgreen fluorescent protein is generated from residues S65-Y66-G67.

[0007] Fluorescent proteins have been used as markers of geneexpression, tracers of cell lineage and as fusion tags to monitorprotein localization within living cells. (M. Chalfie et al., “Greenfluorescent protein as a marker for gene expression,” Science263:802-805; A. B. Cubitt et al., “Understanding, improving and usinggreen fluorescent proteins,” TIBS 20, November 1995, pp. 448-455. U.S.Pat. No. 5,491,084, M. Chalfie and D. Prasher. Furthermore, mutantversions of green fluorescent protein have been identified that exhibitaltered fluorescence characteristics, including altered excitation andemission maxima, as well as excitation and emission spectra of differentshapes. (R. Heim et al., “Wavelength mutations and posttranslationalautoxidation of green fluorescent protein,” Proc. Natl. Acad. Sci. USA,(1994) 91:12501-04; R. Heim et al., “Improved green fluorescence,”Nature (1995) 373:663-665.) These properties add variety and utility tothe arsenal of biologically based fluorescent indicators.

[0008] There is a need for assays of protein phosphorylation that aresimple, sensitive, non-invasive, applicable to living cells and tissuesand that avoid the use of any radioactivity.

SUMMARY OF THE INVENTION

[0009] When fluorescent proteins are modified to incorporate aphosphorylation site recognized by a protein kinase, the fluorescentproteins not only can become phosphorylated by the protein kinase, butthey also can exhibit different fluorescent characteristics in theirun-phosphorylated and phosphorylated forms when irradiated with lighthaving a wavelength within their excitation spectrum. Thischaracteristic makes fluorescent protein substrates particularly usefulfor assaying protein kinase activity in a sample.

[0010] This invention provides methods for determining whether a samplecontains protein kinase activity. The methods involve contacting thesample with a phosphate donor, usually ATP, and a fluorescent proteinsubstrate of the invention; exciting the fluorescent protein substratewith light of an appropriate wavelength; and measuring the amount of afluorescent property that differs in the un-phosphorylated state andphosphorylated state. An amount that is consistent with the presence ofthe fluorescent protein substrate in its phosphorylated state indicatesthe presence of protein kinase activity, and an amount that isconsistent with the presence of the protein substrate in itsun-phosphorylated state indicates the absence of protein kinaseactivity.

[0011] One embodiment of the above method is for determining the amountof protein kinase activity in a sample. In this method, measuring theamount of a fluorescent property in the sample comprises measuring theamount at two or more time points after contacting the sample with aphosphate donor and a fluorescent protein substrate of the invention,and determining the quantity of change or rate of change of the measuredamount. The quantity or rate of change of the measured amount reflectsthe amount of protein kinase activity in the sample.

[0012] In another aspect, the invention provides methods for determiningwhether a cell exhibits protein kinase activity. The methods involve thesteps of providing a transfected host cell of the invention thatproduces a fluorescent protein substrate of the invention; exciting theprotein substrate in the cell with light of an appropriate wavelength;and measuring the amount of a fluorescent property that differs in theun-phosphorylated and phosphorylated states. An amount that isconsistent with the presence of the protein substrate in itsphosphorylated state indicates the presence of protein kinase activity,and an amount that is consistent with the presence of the proteinsubstrate in its un-phosphorylated state indicates the absence ofprotein kinase activity or the presence of phosphatase activity.

[0013] In another aspect, the invention provides methods for determiningthe amount of activity of a protein kinase in one or more cells from anorganism. The methods involve providing a transfected host cellcomprising a recombinant nucleic acid molecule comprising expressioncontrol sequences operatively linked to a nucleic acid sequence codingfor the expression of a fluorescent protein substrate of the invention,the cell expressing the fluorescent protein substrate; exciting theprotein substrate in the cell with light; and measuring the amount of afluorescent property that differs in the un-phosphorylated andphosphorylated states at two or more time points after contacting thesample with a phosphate donor and a fluorescent protein substrate, anddetermining the quantity or rate of change of the measured amount. Thequantity or rate of change of the measured amount reflects the amount ofprotein kinase activity in the sample.

[0014] This invention also provides screening methods for determiningwhether a compound alters the activity of a protein kinase. The methodsinvolve contacting a sample containing a known amount of protein kinaseactivity with the compound, a phosphate donor for the protein kinase anda fluorescent protein substrate of the invention; exciting the proteinsubstrate; measuring the amount of protein kinase activity in the sampleas a function of the quantity or rate of change of a fluorescentproperty that differs in the un-phosphorylated and phosphorylatedstates; and comparing the amount of activity in the sample with astandard activity for the same amount of the protein kinase. Adifference between the amount of protein kinase activity in the sampleand the standard activity indicates that the compound alters theactivity of the protein kinase.

[0015] Another aspect of the drug screening methods involve determiningwhether a compound alters the protein kinase activity in a cell. Themethods involve providing first and second transfected host cellsexhibiting protein kinase activity and expressing a fluorescent proteinsubstrate of the invention; contacting the first cell with an amount ofthe compound; contacting the second cell with a different amount of thecompound; exciting the protein substrate in the first and second cells;measuring the amount of protein kinase activity as a function of thequantity of change or rate of change of a fluorescent property thatdiffers in the un-phosphorylated and phosphorylated states in the firstand second cells; and comparing the amount in the first and secondcells. A difference in the amount indicates that the compound altersprotein kinase activity in the cell.

[0016] This invention also provides fluorescent protein substrates for aprotein kinase. Fluorescent protein substrates for a protein kinasecomprise a fluorescent protein moiety and a phosphorylation site for aprotein kinase. The protein substrate exhibits a different fluorescentproperty in the phosphorylated state than in the unphosphorylated state.In a preferred embodiment, the fluorescent protein is anAequorea-related fluorescent protein. In another embodiment, thephosphorylation site is located within about 5, 10, 15 or 20 amino acidsof a terminus, e.g., the amino-terminus, of the fluorescent proteinmoiety. In another embodiment, the protein substrate comprises thephosphorylation site more than 20 amino acids from a terminal of thefluorescent protein moiety and within the fluorescent protein moiety.The phosphorylation site can be one recognized by, for example, proteinkinase A, a cGMP-dependent protein kinase, protein kinase C,Ca²⁺/calmodulin-dependent protein kinase I, Ca²⁺/calmodulin-dependentprotein kinase II or MAP kinase activated protein kinase type 1.

[0017] This invention also provides nucleic acid molecules coding forthe expression of a fluorescent protein substrate for a protein kinaseof the invention. In one aspect, the nucleic acid molecule is arecombinant nucleic acid molecule comprising expression controlsequences operatively linked to a nucleic acid sequence coding for theexpression of a fluorescent protein substrate for a protein kinase ofthe invention. In another aspect, the invention provides transfectedhost cells transfected with a recombinant nucleic acid moleculecomprising expression control sequences operatively linked to a nucleicacid sequence coding for the expression of a fluorescent proteinsubstrate for a protein kinase of the invention.

[0018] In another aspect, this invention provides collections offluorescent protein candidate substrates comprising at least 10different members, each member comprising a fluorescent protein moietyand a variable peptide moiety around the terminus of the fluorescentprotein moiety.

[0019] In another embodiment, the invention provides collections ofrecombinant nucleic acid molecules comprising at least 10 differentrecombinant nucleic acid molecule members, each member comprisingexpression control sequences operatively linked to nucleic acidsequences coding for the expression of a different fluorescent proteincandidate substrate of the invention. The invention also providescollections of host cells comprising at least 10 different host cellmembers, each member comprising the above recombinant nucleic acidmolecules.

[0020] The collections of cells are useful in determining thespecificity of cellular kinases, from either diseased or normal tissues.The screening methods involve providing a collection of transfected hostcells of the invention; culturing the collection of host cells underconditions for the expression of the fluorescent protein candidatesubstrate; and determining for each of a plurality of members from thecollection whether the member contains a fluorescent protein candidatesubstrate that exhibits a fluorescent property different than thefluorescent property exhibited by the non-phosphorylated candidatesubstrate. The presence of fluorescent protein candidate substrate thatexhibits a fluorescent property different than the fluorescent propertyexhibited by the candidate substrate indicates that the candidatesubstrate possesses a peptide moiety that can be phosphorylated by thekinase present in the host cells.

[0021] This invention also provides kits comprising a fluorescentprotein substrate and a phosphate donor.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 is a flow chart showing the steps in an assay method forprotein kinase activity.

[0023]FIG. 2 depicts molecular events in a cell in altering anddetecting fluorescent properties of a fluorescent protein substrate fora protein kinase.

[0024]FIGS. 3A and 3B depict the nucleotide sequence (SEQ ID NO: 1) anddeduced amino acid sequence (SEQ ID NO:2) of a wild-type Aequorea greenfluorescent protein.

[0025]FIGS. 4A and 4B provide a list of the positions and amino acidchanges made for phosphorylation mutants made more than fifteen aminoacids in the primary sequence from the N-terminus (nucleotide=SEQ IDNO:36 amino acid=SEQ ID NO:37), as compared to FIG. 3. Amino acidsunderlined represent the phosphorylation motif, amino acids in bracketsrepresent wild type sequence at those positions.

[0026]FIG. 5 depicts plasmid pRSET containing a region encoding GFP thatis fused in frame with nucleotides encoding an N-terminal polyhistidinetag (nucleotide SEQ ID NO:38 and SEQ ID NO:40; amino acid=SEQ ID NO:39).

[0027] FIGS. 6A-6E show the fluorescence excitation spectra before andafter phosphorylation of N-terminal phospborylation mutants by proteinkinase A using standard phosphorylation conditions. 6 A: 1MSRRRRSI (SEQID NO:31). 6B: 1MRRRRSII (SEQ ID NO:32). 6C: -1MRRRRSIII (SEQ ID NO:33).6D: -2MRRRRSIIIF (SEQ ID NO:34). 6E: -3MRRRRSIIIIF (SEQ ID NO:35). Inall cases the spectrum after phosphorylation has higher amplitude thanthe spectrum before phosphorylation.

[0028]FIG. 7 depicts an expression vector having expression controlsequences operably linked to sequences coding for the expression ofprotein kinase A catalytic subunit (PKA cat) upstream from sequencescoding for the expression of a fluorescent protein substrate(nucleotide=SEQ ID NO:41 and SEQ ID NO:42).

[0029]FIG. 8 depicts the fluorescence excitation spectrum of 1MRRRRSII(SEQ ID NO:33): S65A, N149K, V163A and 1167T before and afterphosphorylation by protein kinase A using standard phosphorylationconditions. The spectrum after phosphorylation has higher amplitude thanthe spectrum before phosphorylation.

DETAILED DESCRIPTION OF THE INVENTION

[0030] I. Methods for Assaying Samples for Protein Kinases

[0031] Protein kinases add a phosphate residue to the phosphorylationsite of a protein, generally through the hydrolysis of ATP to ADP.Fluorescent protein substrates for protein kinases are useful in assaysto determine the amount of protein kinase activity in a sample. Theassays of this invention take advantage of the fact that phosphorylationof the protein substrate results in a change in a fluorescent propertyof the fluorescent protein. Methods for determining whether a sample haskinase activity involve contacting the sample with a fluorescent proteinsubstrate having a phosphorylation site recognized by the protein kinaseto be assayed and with a phosphate donor under selected test conditions.A phosphate donor is a compound containing a phosphate moiety which thekinase is able to use to phosphorylate the protein substrate. ATP(adenosine-5′-triphosphate) is by far the most common phosphate donor.In certain instances, the sample will contain enough of a phosphatedonor to make this step unnecessary. Then the fluorescent proteinsubstrate is excited with light in its excitation spectrum. If theprotein substrate has been phosphorylated, the substrate will exhibitdifferent fluorescent properties, indicating that the sample containsprotein kinase activity. For example, if the phosphorylated form of theprotein substrate has higher fluorescence than the un-phosphorylatedform, the amount of fluorescence in the sample will increase as afunction of the amount of substrate that has been phosphorylated. If thefluorescent property is a change in the wavelength maximum of emission,the change will be detected as a decrease in fluorescence at thewavelength maximum of the un-phosphorylated substrate and an increase influorescence at the wavelength maximum of the phosphorylated substrate.

[0032] The amount of kinase activity in a sample can be determined bymeasuring the amount of a fluorescent property in the sample at a firsttime and a second time after contact between the sample, the fluorescentprotein substrate and a phosphate donor, and determining the degree ofchange or the rate of change in a fluorescent property. For example, ifphosphorylation results in an increase in fluorescence at the excitationwavelength maximum, the fluorescence of the substrate at this wavelengthcan be determined at two times. The amount of enzyme activity in thesample can be calculated as a function of the difference in thedetermined amount of the property at the two times. For example, theabsolute amount of activity can be calibrated using standards of enzymeactivity determined for certain amounts of enzyme after certain amountsof time. The faster or larger the difference in the amount, the moreenzyme activity must have been present in the sample. The amount of afluorescent property can be determined from any spectral or fluorescencelifetime characteristic of the excited substrate, for example, bydetermining the intensity of the fluorescent signal from the proteinsubstrate or the excited state lifetime of the protein substrate, theratio of the fluorescences at two different excitation wavelengths, theratio of the intensities at two different emission wavelengths, or theexcited lifetime of the protein substrate.

[0033] Fluorescence in a sample is measured using a fluorimeter. Ingeneral, excitation radiation from an excitation source having a firstwavelength, passes through excitation optics. The excitation opticscause the excitation radiation to excite the sample. In response,fluorescent proteins in the sample emit radiation which has a wavelengththat is different from the excitation wavelength. Collection optics thencollect the emission from the sample. The device can include atemperature controller to maintain the sample at a specific temperaturewhile it is being scanned. According to one embodiment, a multi-axistranslation stage moves a microtiter plate holding a plurality ofsamples in order to position different wells to be exposed. Themulti-axis translation stage, temperature controller, auto-focusingfeature, and electronics associated with imaging and data collection canbe managed by an appropriately programmed digital computer. The computeralso can transform the data collected during the assay into anotherformat for presentation. This process can be miniaturized and automatedto enable screening many thousands of compounds.

[0034] Methods of performing assays on fluorescent materials are wellknown in the art and are described in, e.g., Lakowicz, J. R., Principlesof Fluorescence Spectroscopy, New York:Plenum Press (1983); Herman, B.,Resonance energy transfer microscopy, in: Fluorescence Microscopy ofLiving Cells in Culture, Part B, Methods in Cell Biology, vol. 30, ed.Taylor, D. L. & Wang, Y. -L., San Diego: Academic Press (1989), pp.219-243; Turro, N. J., Modern Molecular Photochemistry, Menlo Park:Benjamin/Cummings Publishing Col, Inc. (1978), pp. 296-361.

[0035] Enzymatic assays also can be performed on isolated living cellsin vivo, or from samples derived from organisms transfected to expressthe substrate. Because fluorescent protein substrates can be expressedrecombinantly inside a cell, the amount of enzyme activity in the cellor organism of which it is a part can be determined by determining afluorescent property or changes in a fluorescent property of cells orsamples from the organism.

[0036] In one embodiment, shown in FIG. 2, a cell is transiently orstably transfected with an expression vector 200 encoding a fluorescentprotein substrate containing a phosphorylation site for the enzyme to beassayed. This expression vector optionally includes controllingnucleotide sequences such as promotor or enhancing elements. Theexpression vector expresses the fluorescent protein substrate 210 thatcontains the phosphorylation site 211 for the kinase to be detected. Theenzyme to be assayed may either be intrinsic to the cell or may beintroduced by stable transfection or transient co-transfection withanother expression vector encoding the enzyme and optionally includingcontrolling nucleotide sequences such as promoter or enhancer elements.The fluorescent protein substrate and the enzyme preferably are locatedin the same cellular compartment so that they have more opportunity tocome into contact.

[0037] If the cell possesses a high degree of enzyme activity(K=“kinase” 220), the fluorescent protein substrate will bephosphorylated 230 (PO₄), usually through the hydrolysis of ATP. If thecell does not possess kinase activity, or possesses very little, thecell contains-substantial amounts of un-phosphorylated substrate 240.Upon excitation with light of the appropriate wavelength (hv₁) thephosphorylated substrate will fluoresce light (hv₂). Un-phosphorylatedsubstrate exhibits different fluorescent characteristics upon excitationat the same wavelength, and may, for example, not fluoresce at all, orfluoresce weakly. The amount of the fluorescent property is measuredgenerally using the optics 250 and detector 260 of a fluorimeter.

[0038] If the cell contains phosphatases that compete with the proteinkinases, removing the phosphate from the protein substrate, the level ofenzyme activity in the cell can reach an equilibrium betweenphosphorylated and un-phosphorylated states of the protein substrate,and the fluorescence characteristics will reflect this equilibriumlevel. In one aspect, this method can be used to compare mutant cells toidentify which ones possess greater or lesser ratio of kinase tophosphatase activity. Such cells can be sorted by a fluorescent cellsorter based on fluorescence.

[0039] A contemplated variation of the above assay is to use thecontrolling nucleotide sequences to produce a sudden increase in theexpression of either the fluorescent protein substrate or the enzymebeing assayed, e.g., by inducing expression of the construct. Afluorescent property is monitored at one or more time intervals afterthe onset of increased expression. A high amount of the propertyassociated with phosphorylated state reflects a large amount or highefficiency of the kinase. This kinetic determination has the advantageof minimizing any dependency of the assay on the rates of degradation orloss of the fluorescent protein moieties.

[0040] In another embodiment, the vector may be incorporated into anentire organism by standard transgenic or gene replacement techniques.An expression vector capable of expressing the enzyme optionally may beincorporated into the entire organism by standard transgenic or genereplacement techniques. Then, a sample from the organism containing theprotein substrate is tested. For example, cell or tissue homogenates,individual cells, or samples of body fluids, such as blood, can betested.

[0041] II. Screening Assays

[0042] The enzymatic assays of the invention can be used in drugscreening assays to determine whether a compound alters the activity ofa protein kinase. In one embodiment, the assay is performed on a samplein vitro containing the enzyme. A sample containing a known amount ofenzyme activity is mixed with a substrate of the invention and with atest compound. The amount of the enzyme activity in the sample is thendetermined as above, e.g., by measuring the amount of a fluorescentproperty at a first and second time after contact between the sample,the protein substrate, a phosphate substrate, and the compound. Then theamount of activity per mole of enzyme in the presence of the testcompound is compared with the activity per mole of enzyme in the absenceof the test compound. A difference indicates that the test compoundalters the activity of the enzyme.

[0043] In another embodiment, the ability of a compound to alter kinaseactivity in vivo is determined. In an in vivo assay, cells transfectedwith a expression vector encoding a substrate of the invention areexposed to different amounts of the test compound, and the effect onfluorescence in each cell can be determined. Typically, the differenceis calibrated against standard measurements to yield an absolute amountof kinase activity. A test compound that inhibits or blocks the activityor expression of the kinase can be detected by a relative increase inthe property associated with the un-phosphorylated state. The cell canalso be transfected with an expression vector to co-express the kinaseor an upstream signaling component such as a receptor, and fluorescentsubstrate. This method is useful for detecting signaling to a proteinkinase of interest from an upstream component of the signaling pathway.If a signal from an upstream molecule, e.g., a receptor, is inhibited bya drug activity, then the kinase activity will not be altered frombasal. This provides a method for screening for compounds which affectcellular events (including receptor-ligand binding, protein-proteininteractions or kinase activation) which signal to the target kinase.

[0044] This invention also provides kits containing the fluorescentprotein substrate and a phosphate substrate for the protein kinase. Inone embodiment, the kit has a container holding the fluorescent proteinsubstrate and another container holding the phosphate substrate. Proteinkinases of known activity could be included for use as positive controlsand standards.

[0045] III. Fluorescent Protein Substrates for Protein Kinases

[0046] As used herein, the term “fluorescent property” refers to themolar extinction coefficient at an appropriate excitation wavelength,the fluorescence quantum efficiency, the shape of the excitationspectrum or emission spectrum, the excitation wavelength maximum andemission wavelength maximum, the ratio of excitation amplitudes at twodifferent wavelengths, the ratio of emission amplitudes at two differentwavelengths, the excited state lifetime, or the fluorescence anisotropy.A measurable difference in any one of these properties between thephosphorylated and un-phosphorylated states suffices for the utility ofthe fluorescent protein substrates of the invention in assays for kinaseactivity. A measurable difference can be determined by determining theamount of any quantitative fluorescent property, e.g., the amount offluorescence at a particular wavelength, or the integral of fluorescenceover the emission spectrum. Optimally, the protein substrates areselected to have fluorescent properties that are easily distinguishablein the un-phosphorylated and phosphorylated states.

[0047] Determining ratios of excitation amplitude or emission amplitudeat two different wavelengths (“excitation amplitude ratioing” and“emission amplitude ratioing”, respectively) are particularlyadvantageous because the ratioing process provides an internal referenceand cancels out variations in the absolute brightness of the excitationsource, the sensitivity of the detector, and light scattering orquenching by the sample. Furthermore, if phosphorylation of the proteinsubstrate changes its ratio of excitation or emission amplitudes at twodifferent wavelengths, then such ratios measure the extent ofphosphorylation independent of the absolute quantity of the proteinsubstrate. Some of the fluorescent protein substrates described hereindo exhibit a phosphorylation-induced change in the ratio of excitationamplitudes at two different wavelengths. Even if a fluorescent proteinsubstrate does not exhibit a phosphorylation-induced change inexcitation or emission amplitudes at two wavelengths, cells can beprovided that co-express another fluorescent protein that is notsensitive to phosphorylation and whose excitation or emission spectrumis peaked at wavelengths distinct from those of the phosphorylationsubstrate. Provided that the expression of the two proteins are bothcontrolled by the same nucleotide control sequences, their expressionlevels should be closely linked. Therefore ratioing the excitation oremission amplitude of the phosphorylation substrate at its preferredwavelength to the corresponding excitation or emission amplitude of thephosphorylation-insensitive reference protein at its separate preferredwavelength is an alternative method for canceling out variations in theabsolute quantity of cells or overall level of protein expression.

[0048] A. Fluorescent Proteins

[0049] As used herein, the term “fluorescent protein” refers to anyprotein capable of fluorescence when excited with appropriateelectromagnetic radiation. This includes fluorescent proteins whoseamino acid sequences are either naturally occurring or engineered (i.e.,analogs). Many cnidarians use green fluorescent proteins (“GFPs”) asenergy-transfer acceptors in bioluminescence. A “green fluorescentprotein,” as used herein, is a protein that fluoresces green light.Similarly, “blue fluorescent proteins” fluoresce blue light and “redfluorescent proteins” fluoresce red light. GFPs have been isolated fromthe Pacific Northwest jellyfish, Aequorea Victoria, the sea pansy,Renilla reniformis, and Phialidium gregarium. W. W. Ward et al.,Photochem. Photobiol., 35:803-808 (1982); L. D. Levine et al., Comp.Biochem. Physiol., 72B:77-85 (1982).

[0050] A variety of Aequorea-related fluorescent proteins having usefulexcitation and emission spectra have been engineered by modifying theamino acid sequence of a naturally occurring GFP from Aequorea victoria.(D.C. Prasher et al., Gene, 111:229-233 (1992); R. Heim et al., Proc.Natl. Acad. Sci., USA, 91:12501-04 (1994); U.S. patent application Ser.No. 08/337,915, filed Nov. 10, 1994; International applicationPCT/US95/14692, filed Nov. 10, 1995.)

[0051] As used herein, a fluorescent protein is an “Aequorea-relatedfluorescent protein” if any contiguous sequence of 150 amino acids ofthe fluorescent protein has at least 85% sequence identity with an aminoacid sequence, either contiguous or non-contiguous, from the 238amino-acid wild-type Aequorea green fluorescent protein of FIG. 3 (SEQID NO:2). More preferably, a fluorescent protein is an Aequorea-relatedfluorescent protein if any contiguous sequence of 200 amino acids of thefluorescent protein has at least 95% sequence identity with an aminoacid sequence, either contiguous or non-contiguous, from the wild typeAequorea green fluorescent protein of FIG. 3 (SEQ ID NO:2). Similarly,the fluorescent protein may be related to Renilla or Phialidiumwild-type fluorescent proteins using the same standards.

[0052] Optimal alignment of sequences for aligning a comparison windowmay be conducted by the local homology algorithm of Smith and Waterman(1981) Adv. Appl. Math., 2:482, by the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol., 48:443, by the search forsimilarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci.,U.S.A., 85:2444, by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by inspection. The best alignment (i.e., resulting in thehighest percentage of homology over the comparison window, i.e., 150 or200 amino acids) generated by the various methods is selected.

[0053] The percentage of sequence identity is calculated by comparingtwo optimally aligned sequences over the window of comparison,determining the number of positions at which the identical amino acidoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison (i.e., the window size), andmultiplying the result by 100 to yield the percentage of sequenceidentity.

[0054] Aequorea-related fluorescent proteins include, for example andwithout limitation, wild-type (native) Aequorea victoria GFP (D.C.Prasher et al., “Primary structure of the Aequorea victoria greenfluorescent protein,” Gene, (1992) 111:229-33), whose nucleotidesequence (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO:2)are presented in FIG. 3, allelic variants of this sequence, e.g., Q80R,which has the glutamine residue at position 80 substituted with arginine(M. Chalfie et al., Science, (1994) 263:802-805), those Aequorea-relatedengineered versions described in Table I, variants that include one ormore folding mutations and fragments of these proteins that arefluorescent, such as Aequorea green fluorescent protein from which thetwo amino-terminal amino acids have been removed. Several of thesecontain different aromatic amino acids within the central chromophoreand fluoresce at a distinctly shorter wavelength than wild type species.For example, mutants P4 and P4-3 contain (in addition to othermutations) the substitution Y66H, whereas W2 and W7 contain (in additionto other mutations) Y66W. Other mutations both close to the chromophoreregion of the protein and remote from it in primary sequence may affectthe spectral properties of GFP and are listed in the first part of thetable below. TABLE I Extinct. Excitation Emission Coeff. Quantum CloneMutation(s) max (nm) max (nm) (M⁻¹ cm⁻¹) yield Wild none 395 (475) 50821,000 0.77 type (7,150) P4 Y66H 383 447 13,500 0.21 P4-3 Y66H 381 44514,000 0.38 Y145F W7 Y66W 433 (453) 475 (501) 18,000 0.67 N1461 (17,100)M153T V163A N212K W2 Y66W 432 (453) 480 10,000 0.72 I123V  (9,600) Y145HH148R M153T V163A N212K S65T S65T 489 511 39,200 0.68 P4-1 S65T 504(396) 514 14,500 0.53 M153A  (8,600) K238E S65A S65A 471 504 S65C S65C479 507 S65L S65L 484 510 Y66F Y66F 360 442 Y66W Y66W 458 480

[0055] Additional mutations in Aequorea-related fluorescent proteins,referred to as “folding mutations,” improve the ability of GFP to foldat higher temperatures, and to be more fluorescent when expressed inmammalian cells, but have little or no effect on the peak wavelengths ofexcitation and emission. It should be noted that these may be combinedwith mutations that influence the spectral properties of GFP to produceproteins with altered spectral and folding properties. Folding mutationsinclude: T44A, F64L, V68L, S72A, F99S, Y145F, N1461, M153T or A, V163A,1167T, S175G, S205T and N212K.

[0056] This invention contemplates the use of other fluorescent proteinsin fluorescent protein substrates for protein kinases. The cloning andexpression of yellow fluorescent protein from Vibrio fischeri strain Y-1has been described by T. O. Baldwin et al., Biochemistry (1990)29:5509-15. This protein requires flavins as fluorescent co-factors. Thecloning of Peridinin-chlorophyll a binding protein from thedinoflagellate Symbiodinium sp. was described by B. J. Morris et al.,Plant Molecular Biology, (1994) 24:673:77. One useful aspect of thisprotein is that it fluoresces red. The cloning of phycobiliproteins frommarine cyanobacteria such as Synechococcus, e.g., phycoerythrin andphycocyanin, is described in S. M. Wilbanks et al., J. Biol. Chem.(1993) 268:1226-35. These proteins require phycobilins as fluorescentco-factors, whose insertion into the proteins involves auxiliaryenzymes. The proteins fluoresce at yellow to red wavelengths.

[0057] As used herein, the “fluorescent protein moiety” of a fluorescentprotein substrate is that portion of the amino acid sequence of afluorescent protein substrate which, when the amino acid sequence of thefluorescent protein substrate is optimally aligned with the amino acidsequence of a naturally occurring fluorescent protein, lies between theamino terminal and carboxy terminal amino acids, inclusive, of the aminoacid sequence of the naturally occurring fluorescent protein.

[0058] It has been found that fluorescent proteins can be geneticallyfused to other target proteins and used as markers to identify thelocation and amount of the target protein produced. Accordingly, thisinvention provides fusion proteins comprising a fluorescent proteinmoiety and additional amino acid sequences. Such sequences can be, forexample, up to about 15, up to about 50, up to about 150 or up to about1000 amino acids long. The fusion proteins possess the ability tofluoresce when excited by electromagnetic radiation. In one embodiment,the fusion protein comprises a polyhistidine tag to aid in purificationof the protein.

[0059] B. Phosphorylation Sites For Protein Kinases

[0060] Fluorescent protein substrates for a protein kinase are thesubset of fluorescent proteins as defined above whose amino acidsequence includes a phosphorylation site. Fluorescent protein substratescan be made by modifying the amino acid sequence of an existingfluorescent protein to include a phosphorylation site for a proteinkinase. Fluorescent protein substrates for protein kinases are not meantto include naturally occurring fluorescent proteins or currently knownmutant fluorescent proteins. Such previously known fluorescent proteinsor mutants may be substrates for protein kinases, but do not exhibit anydetectable change in fluorescent properties upon phosphorylation.

[0061] As used herein, the term “phosphorylation site for a proteinkinase” refers to an amino acid sequence which, as part of apolypeptide, is recognized by a protein kinase for the attachment of aphosphate moiety. The phosphorylation site can be a site recognized by,for example, protein kinase A, a cGMP-dependent protein kinase, proteinkinase C, Ca²⁺/calmodulin-dependent protein kinase I,Ca²⁺/calmodulin-dependent protein kinase II or MAP kinase activatedprotein kinase type 1.

[0062] The preferred consensus sequence for protein kinase A is RRXSZ(SEQ ID NO:3) or RRXTZ (SEQ ID NO:4), wherein X is any amino acid and Zis a hydrophobic amino acid, preferably valine, leucine or isoleucine.Many variations in the above sequence are allowed, but generally exhibitpoorer kinetics. For example, lysine (K) can be substituted for thesecond arginine. Many consensus sequences for other protein kinases havebeen tabulated, e.g. by Kemp, B. E. and Pearson, R. B. (1990) TrendsBiochem. Sci. 15: 342-346; Songyang, Z. et al. (1994) Current Biology 4:973-982.

[0063] For example, a fluorescent protein substrate selective forphosphorylation by cGMP-dependent protein kinase can include thefollowing consensus sequence: BKISASEFDR PLR (SEQ ID NO:5), where Brepresents either lysine (K) or arginine (R), and the first S is thesite of phosphorylation (Colbran et al. (1992) J. Biol. Chem. 267:9589-9594). The residues DRPLR (SEQ ID NO:6) are less critical than thephenylalanine (F) just preceding them for specific recognition bycGMP-dependent protein kinase in preference to cAMP-dependent proteinkinase.

[0064] Either synthetic or naturally occurring motifs can be used tocreate a protein kinase phosphorylation site. For example, peptidesincluding the motif XRXXSXRX (SEQ ID NO:7), wherein X is any amino acid,are among the best synthetic substrates (Kemp and Pearson, supra) forprotein kinase C. Alternatively, the Myristoylated Alanine-Rich Kinase Csubstrate (“MARCKS”) is one of the best substrates for PKC and is a realtarget for the kinase in vivo. The sequence around the phosphorylationsite of MARCKS is KKKKRFSFK (SEQ ID NO:8) (Graff et al. (1991) J. Biol.Chem. 266:14390-14398). Either of these two sequences can beincorporated into a fluorescent protein to make it a substrate forprotein kinase C.

[0065] A protein substrate for Ca²⁺/calmodulin-dependent protein kinaseI is derived from the sequence of synapsin I, a known optimal substratefor this kinase. The recognition sequence around the phosphorylationsite is LRRLSDSNF (SEQ ID NO:9) (Lee et al. (1994) Proc. Natl. Acad.Sci. USA 91:6413-6417).

[0066] A protein substrate selective for Ca²⁺/calmodulin-dependentprotein kinase II is derived from the sequence of glycogen synthase, aknown optimal substrate for this kinase. The recognition sequence aroundthe phosphorylation site is KKLNRTLTVA (SEQ ID NO: 10) (Stokoe et al.(1993) Biochem. J. 296:843-849). A small change in this sequence toKKANRTLSVA (SEQ ID NO: 11) makes the latter specific for MAP kinaseactivated protein kinase type 1.

[0067] In one embodiment, the fluorescent protein substrate contains aphosphorylation site around one of the termini, in particular, theamino-terminus, of the fluorescent protein moiety. The site preferablyis located in a position within five, ten, fifteen, or twenty aminoacids of a position corresponding to the wild type amino-terminal aminoacid of the fluorescent protein moiety (“within twenty amino acids ofthe amino-terminus”). This includes sites engineered into the existingamino acid sequence of the fluorescent protein moiety and sites producesby extending the amino terminus of the fluorescent protein moiety.

[0068] One may, for example, modify the existing sequence of wild typeAequorea GFP or a variant or it as listed above to include aphosphorylation site within the first ten or twenty amino acids. In oneembodiment, the naturally occurring sequence is modified as follows:wild type: MSKGEELFTG (SEQ ID NO:43) substrate: MRRRRSIITG (SEQ IDNO:12).

[0069] One may include modifying the naturally occurring sequence ofAequorea GFP by introducing a phosphorylation site into an extendedamino acid sequence of such a protein created by adding flankingsequences to the amino terminus, for example: wild type: MSKGEELFTG (SEQID NO:43) substrate: MRRRRSIIIIFTG (SEQ ID NO:13).

[0070] Fluorescent protein substrates having a phosphorylation sitearound a terminus of the fluorescent protein moiety offer the followingadvantages. First, it is often desirable to append additional amino acidresidues onto the fluorescent protein moiety in order to create aspecific phosphorylation consensus sequence. Such a sequence is muchless likely to disrupt the folding pattern of the fluorescent proteinwhen appended onto the terminus than when inserted into the interior ofthe protein sequence. Second, different phosphorylation motifs can beinterchanged without significant disruption of GFP therefore providing ageneral method of measuring different kinases. Third, thephosphorylation site is exposed to the surface of the protein and,therefore, more accessible to protein kinases. Fourth, we havediscovered that phosphorylation at sites close to the N-terminus of GFPcan provide large changes in fluorescent properties if the site ofphosphorylation is chosen such that the Ser or Thr residue which isphosphorylated occupies a position which in the wild-type protein wasoriginally negatively or positively charged. Specifically, replacementof Glu 6 by a non-charged Ser or Thr residue can significantly disruptfluorescence of GFP when made within the right context of surroundingamino acids. Phosphorylation of the serine or threonine will restorenegative charge to this position and thereby increases fluorescence.

[0071] In another embodiment, the fluorescent protein substrate includesa phosphorylation site remote from the terminus, e.g., that is separatedby more than about twenty amino acids from the terminus of thefluorescent protein moiety and within the fluorescent protein moiety.One embodiment of this form includes the Aequorea-related fluorescentprotein substrate comprising the substitution H217S, creating aconsensus protein kinase A phosphorylation site. Additionally,phosphorylation sites comprising the following alterations based on thesequence of wild type Aequorea GFP exhibit fluorescent changes uponphosphorylation: 69RRFSA (SEQ ID NO:14) and 214KRDSM (SEQ ID NO:15).

[0072] The practitioner should consider the following in selecting aminoacids for substitution within the fluorescent protein moiety remote inprimary amino acid sequence from the terminus. First, it is preferableto select amino acid sequences within the fluorescent protein moietythat resemble the sequence of the phosphorylation site. In this way,fewer amino acid substitutions in the native protein are needed tointroduce the phosphorylation site into the fluorescent protein. Forexample, protein kinase A recognizes the sequence RRXSZ (SEQ ID NO:46)or RRXTZ (SEQ ID NO:47), wherein X is any amino acid and Z is ahydrophobic amino acid. Serine or threonine is the site ofphosphorylation. It is preferable to introduce this sequence into thefluorescent protein moiety at sequences already containing Ser or Thr,so that Ser or Thr are not substituted in the protein. More preferablythe phosphorylation site is created at locations having some existinghomology to the sequence recognized by protein kinase A, e.g., having aproximal Arg or hydrophobic residues with the same spatial relationshipas in the phosphorylation site.

[0073] Second, locations on the surface of the fluorescent protein arepreferred for phosphorylation sites. This is because surface locationsare more likely to be accessible to protein kinases than interiorlocations. Surface locations can be identified by computer modeling ofthe fluorescent protein structure or by reference to the crystalstructure of Aequorea GFP. Also, charged amino acids in the fluorescentprotein are more likely to lie on the surface than inside thefluorescent protein, because such amino acids are more likely to beexposed to water in the environment.

[0074] In cases where the phosphorylation site is either at theN-terminus or remote from it, the amino acid context around thephosphorylation site needs to be optimized in order to maximize thechange in fluorescence. Amino acid substitutions that change large bulkyand or hydrophobic amino acids to smaller and less hydrophobicreplacements are generally helpful. Similarly large charged amino acidscan be replaced by smaller, less charged amino acids. For example:

[0075] a/Hydrophobic to less hydrophobic

[0076] Phe to Leu

[0077] Leu to Ala

[0078] b/Charged to charged but smaller

[0079] Glu to Asp

[0080] Arg to Lys

[0081] c/Charged to less charged

[0082] Glu to Gln

[0083] Asp to Asn

[0084] d/Charged to polar

[0085] Glu to Thr

[0086] Asp to Ser

[0087] e/Charged to non-polar

[0088] Glu to Leu

[0089] Asp to Ala

[0090] These changes can be accomplished by directed means or usingrandom iterative approaches where changes are made randomly and the bestones selected based upon their change in fluorescent properties afterphosphorylation by an appropriate kinase.

[0091] Third, amino acids at distant locations from the actual site ofphosphorylation can be varied to enhance fluorescence changes uponphosphorylation. These mutations can be created through site directedmutagenesis, or through random mutagenesis, for example by error-pronePCR, to identify mutations that enhance either absolute fluorescence orthe change in fluorescence upon phosphorylation. The identification ofmutants remote in primary sequence from the N-terminus identifiespotentially interacting sequences which may provide additional areas inwhich further mutagenesis could be used to refine the change influorescence upon phosphorylation. For example, it has been determinedthat mutations around the amino terminus phosphorylation site interact(either transiently during folding, or in a stable fashion) with aminoacids at positions 171 and 172, and that point mutations thatsignificantly disrupt fluorescence of GFP by changing negative topositive charges near the amino terminus can be rescued by changing apositive to a negative charge at position 171.

[0092] In the phosphorylation mutant 50 the sequence is a/ and forreference the wild type sequence b/ is listed below. a/ MSKRRDSLT (SEQID NO:16) b/ MSKGEELFT (SEQ ID NO:44)

[0093] The phosphorylation mutant has only 7% of the fluorescence ofwild type protein. However, its fluorescence can be restored to 80% ofwild type by 2 amino acid changes, E171K and I172V, positions which arequite remote in linear sequence from the amino terminus.

[0094] Thus, changes in charge at E171K (negative to positive) canalmost completely restore the fluorescence of the phosphorylationmutant, strongly suggesting that the original loss of fluorescence aroseprimarily through changes in charge caused by the point mutations. It isclear that the addition and loss of charge at positions around, and atthe phosphorylation site, have a significant impact on fluorescenceformation. The fact that charge alone can significantly affect thefluorescence properties of GFP is highly significant within the scope ofthe present application since phosphorylation involves the addition of 2negative charges associated with the phosphate group (OPO₃ ⁻²) on theserine residue.

[0095] In the above case the mutations restore fluorescence of thephosphorylation mutant, without significantly increasing the magnitudeof the change in fluorescence upon phosphorylation. Nevertheless theidentification of these positions in GFP provides a valuable tool tofurther optimize changes in fluorescence upon phosphorylation bycreating random mutations at codons around positions 171, 172 and 173 toidentify mutations that enhance changes in fluorescence uponphosphorylation.

[0096] This can be achieved by co-expressing the kinase of interest withthe fluorescent substrate of the invention containing random mutationswhich may enhance the fluorescence changes upon phosphorylation inbacteria (in the example above these would be NNK mutations at codons171, 172 and 173, where N represents a random choice of any of the fourbases and K represents a random choice of guanine or thymine). Theexpression vector containing the mutated fluorescent substrates and thekinase are transformed into host bacteria and the individual bacterialcolonies grown up. Each colony is derived from a single cell, and hencecontains a single unique mutant fluorescent substrate grown up.

[0097] The individual colonies may then be grown up and screened forfluorescence either by fluorescence activated cell sorting (FACS), or byobservation under a microscope. Those that exhibit the greatestfluorescence can then be rescreened under conditions in which the kinasegene is inactivated. This can be achieved by appropriate digests of thekinase gene by restriction enzymes that specifically cut within thekinase but not GFP. Comparison of the brightness of the mutant first inthe presence of kinase then in its absence indicates the relative effectof phosphorylation on the mutant GFP.

[0098] C. Production of Fluorescent Protein Substrates for ProteinKinases

[0099] While certain fluorescent protein substrates for protein kinasescan be prepared chemically, for example, by coupling a peptide moiety tothe amino terminus of a fluorescent protein, it is preferable producefluorescent protein substrates recombinantly.

[0100] Recombinant production of a fluorescent protein substrateinvolves expressing a nucleic acid molecule having sequences that encodethe protein. As used herein, the term “nucleic acid molecule” includesboth DNA and RNA molecules. It will be understood that when a nucleicacid molecule is said to have a DNA sequence, this also includes RNAmolecules having the corresponding RNA sequence in which “U” replaces“T.” The term “recombinant nucleic acid molecule” refers to a nucleicacid molecule which is not naturally occurring, and which comprises twonucleotide sequences which are not naturally joined together.Recombinant nucleic acid molecules are produced by artificialcombination, e.g., genetic engineering techniques or chemical synthesis.

[0101] In one embodiment, the nucleic acid encodes a fusion protein inwhich a single polypeptide includes the fluorescent protein moietywithin a longer polypeptide. In another embodiment the nucleic acidencodes the amino acid sequence of consisting essentially of afluorescent protein modified to include a phosphorylation site. Ineither case, nucleic acids that encode fluorescent proteins are usefulas starting materials.

[0102] Nucleic acids encoding fluorescent proteins can be obtained bymethods known in the art. For example, a nucleic acid encoding a greenfluorescent protein can be isolated by polymerase chain reaction of cDNAfrom A. victoria using primers based on the DNA sequence of A. victoriagreen fluorescent protein, as presented in FIG. 3. PCR methods aredescribed in, for example, U.S. Pat. No. 4,683,195; Mullis et al. (1987)Cold Spring Harbor Symp. Quant. Biol. 51:263; and Erlich, ed., PCRTechnology, (Stockton Press, NY, 1989).

[0103] Mutant versions of fluorescent proteins can be made bysite-specific mutagenesis of other nucleic acids encoding fluorescentproteins, or by random mutagenesis caused by increasing the error rateof PCR of the original polynucleotide with 0.1 mM MnCl₂ and unbalancednucleotide concentrations. See, e.g., U.S. patent application Ser. No.08/337,915, filed Nov. 10, 1994 or International applicationPCT/US95/14692, filed Nov. 10, 1995.

[0104] Nucleic acids encoding fluorescent protein substrates which arefusions between a polypeptide including a phosphorylation site and afluorescent protein and can be made by ligating nucleic acids thatencode each of these. Nucleic acids encoding fluorescent proteinsubstrates which include the amino acid sequence of a fluorescentprotein in which one or more amino acids in the amino acid sequence of afluorescent protein are substituted to create a phosphorylation site canbe created by, for example, site specific mutagenesis of a nucleic acidencoding a fluorescent protein.

[0105] The construction of expression vectors and the expression ofgenes in transfected cells involves the use of molecular cloningtechniques also well known in the art. Sambrook et al., MolecularCloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., (1989) and Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., (Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc.

[0106] Nucleic acids used to transfect cells with sequences coding forexpression of the polypeptide of interest generally will be in the formof an expression vector including expression control sequencesoperatively linked to a nucleotide sequence coding for expression of thepolypeptide. As used, the term nucleotide sequence “coding forexpression of” a polypeptide refers to a sequence that, upontranscription and translation of mRNA, produces the polypeptide. As anyperson skilled in the art recognizes, this includes all degeneratenucleic acid sequences encoding the same amino acid sequence. This caninclude sequences containing, e.g., introns. As used herein, the term“expression control sequences” refers to nucleic acid sequences thatregulate the expression of a nucleic acid sequence to which it isoperatively linked. Expression control sequences are “operativelylinked” to a nucleic acid sequence when the expression control sequencescontrol and regulate the transcription and, as appropriate, translationof the nucleic acid sequence. Thus, expression control sequences caninclude appropriate promoters, enhancers, transcription terminators, astart codon (i.e., ATG) in front of a protein-encoding gene, splicingsignals for introns, maintenance of the correct reading frame of thatgene to permit proper translation of the MRNA, and stop codons.

[0107] The recombinant nucleic acid can be incorporated into anexpression vector comprising expression control sequences operativelylinked to the recombinant nucleic acid. The expression vector can beadapted for function in prokaryotes or eukaryotes by inclusion ofappropriate promoters, replication sequences, markers, etc.

[0108] The expression vector can be transfected into a host cell forexpression of the recombinant nucleic acid. Host cells can be selectedfor high levels of expression in order to purify the protein. E. coli isuseful for this purpose. Alternatively, the host cell can be aprokaryotic or eukaryotic cell selected to study the activity of anenzyme produced by the cell. The cell can be, e.g., a cultured cell or acell in vivo.

[0109] Recombinant fluorescent protein substrates can be produced byexpression of nucleic acid encoding for the protein in E. coli.Aequorea-related fluorescent proteins are best expressed by cellscultured between about 15° C. and 30° C. but higher temperatures (e.g.37° C.) are possible. After synthesis, these enzymes are stable athigher temperatures (e.g., 37° C.) and can be used in assays at thosetemperatures.

[0110] The construct can also contain a tag to simplify isolation of thesubstrate. For example, a polyhistidine tag of, e.g., six histidineresidues, can be incorporated at the amino or carboxyl terminal of thefluorescent protein substrate. The polyhistidine tag allows convenientisolation of the protein in a single step by nickel-chelatechromatography.

[0111] Alternatively, the substrates need not be isolated from the hostcells. This method is particularly advantageous for the assaying for thepresence of protein kinase activity in situ.

[0112] IV. Libraries of Candidate Substrates

[0113] The inclusion of a phosphorylation site around the amino terminusof a fluorescent protein moiety can provide a fluorescent protein that,when phosphorylated, can alter a fluorescent property of the protein.Accordingly, this invention provides libraries of fluorescent proteincandidate substrates useful for screening in the identification andcharacterization of sequences that can be recognized and efficientlyphosphorylated by a kinase. Libraries of these proteins can be screenedto identify sequences that can be phosphorylated by kinases of unknownsubstrate specificity, or to characterize differences in kinase activityin, or from, diseased and normal cells or tissues.

[0114] As used herein, a “library” refers to a collection containing atleast 10 different members. Each member of a fluorescent proteincandidate substrate library comprises a fluorescent protein moiety and avariable peptide moiety, which is preferably located near theamino-terminus of the fluorescent protein moiety and preferably hasfewer than about 15 amino acids. The variety of amino acid sequences forthe peptide moiety is at the discretion of the practitioner. Forexample, the library can contain a quite diverse collection of variablepeptide moieties in which most or all of the amino acid positions aresubjected to a non-zero but low probability of substitution. Also, thelibrary can contain variable peptide moieties having an amino acidsequence in which only a few, e.g., one to ten, amino acid positions arevaried, but the probability of substitution at each position isrelatively high.

[0115] Preferably, libraries of fluorescent protein candidate substratesare created by expressing protein from libraries of recombinant nucleicacid molecules having expression control sequences operatively linked tonucleic acid sequences that code for the expression of differentfluorescent protein candidate substrates. Methods of making nucleic acidmolecules encoding a diverse collection of peptides are described in,for example, U.S. Pat. No. 5,432,018 (Dower et al.), U.S. Pat. No.5,223,409 (Ladner et al.), U.S. Pat. No. 5,264,563 (Huse), andInternational patent publication WO 92/06176 (Huse et al.). Forexpression of fluorescent protein candidate substrates, recombinantnucleic acid molecules are used to transfect cells, such that each cellcontains a member of the library. This produces, in turn, a library ofhost cells capable of expressing the library of different fluorescentprotein candidate substrates. The library of host cells is useful in thescreening methods of this invention.

[0116] In one method of creating such a library, a diverse collection ofoligonucleotides having preferably random codon sequences are combinedto create polynucleotides encoding peptides having a desired number ofamino acids. The oligonucleotides preferably are prepared by chemicalsynthesis. The polynucleotides encoding variable peptide moiety can thenbe coupled to the 5′ end of a nucleic acid coding for the expression ofa fluorescent protein moiety or a carboxy-terminal portion of it. Thatis, the fluorescent protein moiety can be cut back to eliminate up to 20amino acids of the reference fluorescent protein. This creates arecombinant nucleic acid molecule coding for the expression of afluorescent protein candidate substrate having a peptide moiety fused tothe amino terminus of the fluorescent protein. This recombinant nucleicacid molecule is then inserted into an expression vector to create arecombinant nucleic acid molecule comprising expression controlsequences operatively linked to the sequences encoding the candidatesubstrate.

[0117] To generate the collection of oligonucleotides which forms aseries of codons encoding a random collection of amino acids and whichis ultimately cloned into the vector, a codon motif is used, such as(NNK)_(x), where N may be A, C, G, or T (nominally equimolar), K is G orT (nominally equimolar), and x is the desired number of amino acids inthe peptide moiety, e.g., 15 to produce a library of 15-mer peptides.The third position may also be G or C, designated “S”. Thus, NNK or NNS(i) code for all the amino acids, (ii) code for only one stop codon, and(iii) reduce the range of codon bias from 6:1 to 3:1. The expression ofpeptides from randomly generated mixtures of oligonucleotides inappropriate recombinant vectors is discussed in Oliphant et al., Gene44:177-183 (1986).

[0118] An exemplified codon motif (NNK)₆ (SEQ ID NO: 17) produces 32codons, one for each of 12 amino acids, two for each of five aminoacids, three for each of three amino acids and one (amber) stop codon.Although this motif produces a codon distribution as equitable asavailable with standard methods of oligonucleotide synthesis, it resultsin a bias against peptides containing one-codon residues.

[0119] An alternative approach to minimize the bias against one-codonresidues involves the synthesis of 20 activated tri-nucleotides, eachrepresenting the codon for one of the 20 genetically encoded aminoacids. These are synthesized by conventional means, removed from thesupport but maintaining the base and 5-HO-protecting groups, andactivating by the addition of 3′O-phosphoramidite (and phosphateprotection with beta-cyanoethyl groups) by the method used for theactivation of mononucleosides, as generally described in McBride andCaruthers, Tetrahedron Letters 22:245 (1983). Degenerate “oligocodons”are prepared using these trimers as building blocks. The trimers aremixed at the desired molar ratios and installed in the synthesizer. Theratios will usually be approximately equimolar, but may be a controlledunequal ratio to obtain the over- to under-representation of certainamino acids coded for by the degenerate oligonucleotide collection. Thecondensation of the trimers to form the oligocodons is done essentiallyas described for conventional synthesis employing activatedmononucleosides as building blocks. See generally, Atkinson and Smith,Oligonucleotide Synthesis, M. J. Gait, ed. p35-82 (1984). Thus, thisprocedure generates a population of oligonucleotides for cloning that iscapable of encoding an equal distribution (or a controlled unequaldistribution) of the possible peptide sequences.

[0120] Libraries of amino terminal phosphorylation sites may also beannealed to libraries of randomly mutated GFP sequences to enable theselection of optimally responding substrates.

[0121] V. Methods for Screening Libraries of Candidate Substrates

[0122] Libraries of host cells expressing fluorescent protein candidatesubstrates are useful in identifying fluorescent proteins having peptidemoieties that alter a fluorescent property of the fluorescent protein.Several methods of using the libraries are envisioned. In general, onebegins with a library of recombinant host cells, each of which expressesa different fluorescent protein candidate substrate. Each cell isexpanded into a clonal population that is genetically homogeneous.

[0123] In a first method, the desired fluorescent property is measuredfrom each clonal population before and at least one specified time aftera known change in intracellular protein kinase activity. This change inkinase activity could be produced by transfection with a gene encodingthe kinase, by induction of kinase gene expression using expressioncontrol elements, or by any condition that post-translationallymodulates activity of a kinase that has already been expressed. Examplesof the latter include cell surface receptor mediated elevation ofintracellular cAMP to activate cAMP-dependent surface receptor mediatedincreases of intracellular cGMP to activate cGMP-dependent proteinkinase, cytosolic free calcium to activate Ca²⁺/calmodulin-dependentprotein kinase types I, II, or IV, or the production of diacylglycerolto activate protein kinase C, etc. One then selects for the clone(s)that show the biggest or fastest change in the desired fluorescenceproperty. This method detects fluorescent protein mutants whose foldingand maturation was influenced by phosphorylation as well as thoseaffected by phosphorylation after maturation.

[0124] One embodiment of this method exploits the fact that thecatalytic subunit of cAMP-dependent protein kinase is constitutivelyactive in the absence of the regulatory subunit and is growth-inhibitoryin E. coli and most mammalian cells. Therefore, the cells tend to shedthe kinase gene by recombination. The change in kinase activity isobtained by culturing the cells for a time sufficient to lose the kinasegene.

[0125] In a second method the host cells do not express the proteinkinase of interest. Each clonal population is separately lysed. ATP isthen added to the lysate. After an incubation period to allowphosphorylation by background kinases, the fluorescence property ismeasured. Then exogenous protein kinase is added to the lysate and thefluorescent property is re-measured at one or more specified timepoints. Again one selects for the clone(s) that show the biggest orfastest change(s) in the desired fluorescence property. Because littleor no fresh protein synthesis is likely to occur in the lysate, thismethod would discriminate against mutants which are sensitive tophosphorylation only during their folding and maturation.

[0126] In one embodiment of this method, the lysate is split into twoaliquots, one of which is mixed with kinase and ATP, the other of whichreceives only ATP. One selects for the clone(s) that show the biggestdifference in fluorescence property between the two aliquots.

[0127] The nucleic acids from cells exhibiting the different propertiescan be isolated from the cells. Candidate substrates having differentfluorescent properties can be tested further to identify the source ofthe difference.

[0128] The host cell also can be transfected with an expression vectorcapable of expressing an enzyme, such as a protein kinase, whose effecton the fluorescent property is to be tested.

EXAMPLES

[0129] A. Phosphorylation Sites Located in the Amino Acid Sequence ofAeguorea GFP Remote in the Primary Amino Acid Sequence from theN-terminus

[0130] Potential sites for phosphorylation were chosen at or close topositions in GFP which had previously been identified to exertsignificant effects on fluorescence, or which had a higher probabilityof surface exposure based on computer algorithms (FIG. 4). For example,in a mutant called H9, Ser202 and Thr203 are mutated to F and Irespectively, creating a large change in spectral properties (see alsoEhrig et al, 1995). Therefore in one mutant, 199RRLSI (SEQ ID NO: 18), apotential site of phosphorylation was created around Ser202, whosephosphorylation should significantly affect the fluorescent properties.Similarly the amino acids located at positions 72 and 175 have beenimplicated in increased folding efficiency of GFP at higher temperaturesand were made into potential sites of phosphorylation in separatemutants.

[0131] A complete list of the positions and amino acid changes made foreach phosphorylation mutant in this series is outlined in FIG. 4. GFPwas expressed in E. coli using the expression plasmid pRSET(Invitrogen), in which the region encoding GFP was fused in frame withnucleotides encoding an N-terminal polyhistidine tag (FIG. 5). Thesequence changes were introduced by site-directed mutagenesis using theBio-Rad mutagenesis kit (Kunkel, T. A. (1985) Proc. Natl. Acad. Sci.82:488-492, Kunkel,T. A., Roberts, J. D., and Zakour, R. A. (1987) MethEnzymol 154:367-382) and confirmed by sequencing. The recombinantproteins were induced with IPTG and expressed in bacteria and purifiedby nickel affinity chromatography. The sequence changes, relativefluorescence, relative rate of phosphorylation and % change influorescence upon phosphorylation are listed in Table II. In those caseswhere the protein exhibited no fluorescence after insertion of thephosphorylation site no determinations were made on the effect ofphosphorylation on fluorescence. TABLE II Relative fluorescence, rate ofphosphorylation and change in fluorescence upon phosphorylation formutants incorporating phosphorylation sites remote from the N-terminusFluorescence % Change in SEQ before Relative fluorescence IDphosphorylation rates of after incubation NO: Sequence (% of wild type)phosphorylation with kinase 19 25RRFSV 95 1.75  −5 20 68RRFSR 0 n.d n.d14 68RRFSA 6 0.6  +10 21 94RRSIF 0 n.d n.d 22 131RRGSIL 0 n.d n.d 23155KRKSGI 86 2.5    0 24 172RRGSV 90 1.57   0 18 199RRLSI 0 n.d n.d 15214KRDSM 21 1.88 +40

[0132] Bold letters indicate site of phosphorylation. Numbers prior tothe sequence indicate amino acid position in wild type GFP (FIG. 3, SEQID NO:2) where phosphorylation site starts. The relative rates ofphosphorylation compare the rate of phosphorylation of the givenphosphorylation site with the endogenous protein kinase Aphosphorylation site in Aequorea GFP (HKFSV (SEQ ID NO:45)) measured byincorporation of ³²P after incubation of the purified substrate andprotein kinase A catalytic subunit in the presence of ³²P-labelled ATPusing 3 μg GFP, 5 μg protein kinase A catalytic subunit for 10 minutesat 30° C. in standard phosphorylation buffer (20 mM MOPS pH 6.5, 100 mMKCl, 100 μM ATP, 3 mM MgCl₂ 1 mM DTT and 100 uCi ³²P-labeled ATP.Reactions were terminated by blotting onto phosphocellulose paper andwashing with 10% phosphoric acid. The % change in fluorescencerepresents the increase in fluorescence (475 nm excitation, 510 nmemission) observed in each purified protein resulting from incubationwith excess protein kinase A catalytic subunit for 1 hour at 30° C.using the same phosphorylation conditions as described above except thatno ³²P-labeled ATP was present and that after the reaction time wascomplete samples were analyzed in the fluorimeter rather than blottedonto phosphocellulose paper.

[0133] The greatest changes in fluorescence occurred in mutant 214KRDSM(SEQ ID NO: 15) which exhibited a 40% change in fluorescence uponphosphorylation. However analysis of the kinetics of phosphorylationusing γ-³²P-labeled ATP demonstrated that the site is poorlyphosphorylated by protein kinase A. Wild type GFP contains a mediocreconsensus phosphorylation site (25HKFSV (SEQ ID NO:45)) that can bephosphorylated by protein kinase A in vitro with relatively slowkinetics. While phosphorylation at this position has no detectableeffect on the fluorescence of GFP, the rate of phosphorylation at thisposition is used as an internal control between experiments to determinethe relative rates of phosphorylation at sites engineered into theprotein by site directed mutagenesis.

[0134] B. Phosphorylation Sites Around the Amino Terminus

[0135] Sites at the N-terminus of GFP were engineered into GFP by PCR.Initial studies attempted to preserve the native sequence as much aspossible. As discussed earlier the positions chosen for phosphorylationwere within the first 5 amino acids of GFP and encompassed all chargedresidues within this region. The sequence changes, relativefluorescence, relative rates of phosphorylation and % change influorescence upon phosphorylation are tabulated in Table III. TABLE IIIRelative fluorescence, rate of phosphorylation and change influorescence upon phosphorylation for phosphorylation sites inserted atthe N-terminus Relative fluorescence Relative SEQ ID as a % of rates of% Change in NO: Sequence wild type phosphorylation fluorescence 481MSKGEELF 100 1.0 0 25 1MRKGSCLF 40 5.1 5.7 26 1MRKGSLLF 52 1.6 8.0 271MRRESLLF 30 3.0 6.0 28 1MRRDSCLF 27 3.7 17 29 1MSRRDSCF 43 2.1 25 301MSKRRDSL 7 5.5 5.1

[0136] Numbers prior to the sequence indicate amino acid position inwild type GFP where phosphorylation site starts. The relative rates ofphosphorylation compare the rate of phosphorylation of the givenphosphorylation site with the endogenous protein kinase Aphosphorylation site in Aequorea GFP (HKFSV (SEQ ID NO:45)) measured byincorporation of ³²p after incubation of the purified substrate andprotein kinase A catalytic subunit in the presence of ³²P-labelled ATPusing the standard protocols described earlier. The % change influorescence represents the change in fluorescence (488 nm excitation,511 nm emission) observed in each purified protein as a result ofincubation with excess protein kinase A catalytic subunit for 1 hour at30° C. using phosphorylation conditions described earlier.

[0137] These results demonstrated that mutants whose sequence closelyresembles the native protein retain considerable fluorescence, displaygood kinetics of phosphorylation, but show relatively small changes influorescence after phosphorylation. To improve the effect ofphosphorylation on fluorescence, amino acids around the phosphorylationsite were mutated to create an optimal phosphorylation sequence even ifit disordered the existing local tertiary structure. Such disruption waspredicted and found to decrease the basal fluorescence of theseconstructs in their non-phosphorylated state (Table IV). TABLE IVRelative fluorescence before phosphorylation and change in fluorescenceupon phosphorylation for more drastically altered phosphorylation sitesinserted at the N-terminus Relative % Change in SEQ fluorescencefluorescence ID as a % of upon NO: Sequence wild-type phosphorylation 481MSKGEELF(=WT) ≅100 0 31 1MSRRRRSI 5.8 40 32 1MRRRRSII 5.1 70 33−1MRRRRSIII n.d. 43 34 −2MRRRRSIIIF 0.7 15 35 −3MRRRRSIIIIF 0.6 70

[0138] Numbers prior to the sequence indicate amino acid position inwild type GFP where phosphorylation site starts. Negative numbersindicate extensions onto the wild-type N-terminus. The % change influorescence represents the change in fluorescence (488 excitation, 511emission) observed in each purified protein resulting from incubationwith excess protein kinase A catalytic subunit for 1 hour at 30° C.using standard phosphorylation conditions described earlier.

[0139] Perhaps because of the reduced basal fluorescence,phosphorylation by protein kinase A produced greater percentageincreases in fluorescence in these constructs than in the moreconservative mutations of Table II. Constructs 1MRRRRSII (SEQ ID NO:32)and -3MRRRRSIIIIF (SEQ ID NO:35) displayed the greatest increases, about70%, in fluorescence upon phosphorylation using the standard conditions,as shown in FIG. 6. However, these increased percentage increases wereobtained at the cost of a reduced ability to fold at higher temperaturesand relatively poor fluorescence even after phosphorylation. To improvethese characteristics, these mutants were further optimized byadditional random mutagenesis with a novel selection procedure.

[0140] C. Further Optimization of N-terminal Phosphorylation Sites byRandom Mutagenesis of the Remainder of GFP

[0141] The two best constructs from above (1MRRRRSII (SEQ ID NO:32) and-3MRRRRSIII IF (SEQ ID NO:35)) were further mutagenized and screened forvariants that are highly fluorescent when phosphorylated, but weaklyfluorescent when non-phosphorylated. The method involved expression of arandomly mutated fluorescent substrate with or without simultaneousco-expression of the constitutively active catalytic subunit of proteinkinase A in bacteria, and screening the individual mutants to determinethose that are highly fluorescent in the presence but not the absence ofthe kinase.

[0142] To enable co-expression of the kinase and potential substrates, anew expression vector with the kinase C subunit upstream from thefluorescent substrate was constructed (FIG. 7). Random mutations wereintroduced into GFP by error-prone PCR and the resulting population ofmutants cloned into the co-expression vector using the appropriaterestriction sites. The expression vector containing the mutatedfluorescent substrates were transformed into host bacteria andindividual bacterial colonies (each derived from a single cell, andhence containing a single unique mutant fluorescent substrate) weregrown up.

[0143] The colonies were screened for fluorescence either byfluorescence-activated cell sorting (FIG. 8) or by observation under amicroscope. Those that exhibited the greatest fluorescence werere-screened under conditions in which the kinase gene was inactivated.This was achieved in either of two ways. In the first method theco-expression vector was isolated and treated with restrictionendonucleases and modifying enzymes (EcoR1, klenow fragment and T4 DNAligase) to cut the kinase gene, add additional bases and relegate theDNA, causing a frame shift and hence inactivating the gene. The treatedand non-treated plasmids were then re-transformed into bacteria andcompared in fluorescence. Alternatively the plasmids were initiallygrown in a RecA⁻ (recombinase A negative) bacterial strain, where thekinase is stable, to screen for brighter mutants in the presence of thekinase. The plasmid DNA was then isolated and re-transformed into astrain of bacteria which is RecA⁺, in which the kinase is unstable andis lost through homologous recombination of the tandomly repeatedribosome binding sites (rbs). The bacteria have a strong tendency toeliminate the kinase C subunit because it slows their multiplication, socells that splice out the kinase by recombination have a large growthadvantage.

[0144] Comparison of the brightness of the mutant first in the presenceof kinase then in its absence indicates the relative effect ofphosphorylation on the mutant GFP fluorescence (after normalizing forGFP expression levels). A library of approximately 2×10⁶ members wasscreened by this approach. Approximately 500 displayed higher levels offluorescence when screened in the presence of the kinase. Afterinactivation of the kinase, one mutant out of the 500 displayed reducedlevels of fluorescence. The increased fluorescence of the remainder ofthe 500 mutants was independent of the presence of the kinase. Thismutant GFP was isolated and sequenced and found to contain the followingmutations compared to wild-type GFP (FIG. 3, SEQ ID NO:2) (in additionto the N-terminal phosphorylation site 1MRRRRSII (SEQ ID NO:32)): S65A,N149K, V163A and I167T.

[0145] To confirm that this mutant was indeed directly sensitive toprotein kinase A phosphorylation and to quantify its responsively, itwas expressed in the absence of kinase. The E. coli were lysed and theprotein purified as described earlier using a nickel affinity column.The protein exhibited high levels of fluorescence when induced at 30° C.but displayed reduced fluorescence when incubated at 37° C. After suchpreincubation (37° C. overnight) and separation of the less fluorescentmaterial by centrifugation, this protein exhibited the largest change influorescence upon phosphorylation yet observed (FIG. 8). The toleranceof this mutant for 37° C. treatment suggests that this mutant issuitable for use in mammalian cells.

[0146] The present invention provides novel assays for protein kinaseactivity involving novel fluorescent protein substrates. While specificexamples have been provided, the above description is illustrative andnot restrictive. Many variations of the invention will become apparentto those skilled in the art upon review of this specification. The scopeof the invention should, therefore, be determined not with reference tothe above description, but instead should be determined with referenceto the appended claims along with their full scope of equivalents.

[0147] All publications and patent documents cited in this applicationare incorporated by reference in their entirety for all purposes to thesame extent as if each individual publication or patent document were soindividually denoted.

1 48 717 base pairs nucleic acid single linear DNA CDS 1..717 /product=“wild-type Aequorea green fluorescent protein (GFP)” 1 ATG AGT AAA GGAGAA GAA CTT TTC ACT GGA GTT GTC CCA ATT CTT GTT 48 Met Ser Lys Gly GluGlu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 GAA TTA GAT GGTGAT GTT AAT GGG CAC AAA TTT TCT GTC AGT GGA GAG 96 Glu Leu Asp Gly AspVal Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 GGT GAA GGT GAT GCAACA TAC GGA AAA CTT ACC CTT AAA TTT ATT TGC 144 Gly Glu Gly Asp Ala ThrTyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 ACT ACT GGA AAA CTA CCTGTT CCA TGG CCA ACA CTT GTC ACT ACT TTC 192 Thr Thr Gly Lys Leu Pro ValPro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 TCT TAT GGT GTT CAA TGC TTTTCA AGA TAC CCA GAT CAT ATG AAA CGG 240 Ser Tyr Gly Val Gln Cys Phe SerArg Tyr Pro Asp His Met Lys Arg 65 70 75 80 CAT GAC TTT TTC AAG AGT GCCATG CCC GAA GGT TAT GTA CAG GAA AGA 288 His Asp Phe Phe Lys Ser Ala MetPro Glu Gly Tyr Val Gln Glu Arg 85 90 95 ACT ATA TTT TTC AAA GAT GAC GGGAAC TAC AAG ACA CGT GCT GAA GTC 336 Thr Ile Phe Phe Lys Asp Asp Gly AsnTyr Lys Thr Arg Ala Glu Val 100 105 110 AAG TTT GAA GGT GAT ACC CTT GTTAAT AGA ATC GAG TTA AAA GGT ATT 384 Lys Phe Glu Gly Asp Thr Leu Val AsnArg Ile Glu Leu Lys Gly Ile 115 120 125 GAT TTT AAA GAA GAT GGA AAC ATTCTT GGA CAC AAA TTG GAA TAC AAC 432 Asp Phe Lys Glu Asp Gly Asn Ile LeuGly His Lys Leu Glu Tyr Asn 130 135 140 TAT AAC TCA CAC AAT GTA TAC ATCATG GCA GAC AAA CAA AAG AAT GGA 480 Tyr Asn Ser His Asn Val Tyr Ile MetAla Asp Lys Gln Lys Asn Gly 145 150 155 160 ATC AAA GTT AAC TTC AAA ATTAGA CAC AAC ATT GAA GAT GGA AGC GTT 528 Ile Lys Val Asn Phe Lys Ile ArgHis Asn Ile Glu Asp Gly Ser Val 165 170 175 CAA CTA GCA GAC CAT TAT CAACAA AAT ACT CCA ATT GGC GAT GGC CCT 576 Gln Leu Ala Asp His Tyr Gln GlnAsn Thr Pro Ile Gly Asp Gly Pro 180 185 190 GTC CTT TTA CCA GAC AAC CATTAC CTG TCC ACA CAA TCT GCC CTT TCG 624 Val Leu Leu Pro Asp Asn His TyrLeu Ser Thr Gln Ser Ala Leu Ser 195 200 205 AAA GAT CCC AAC GAA AAG AGAGAC CAC ATG GTC CTT CTT GAG TTT GTA 672 Lys Asp Pro Asn Glu Lys Arg AspHis Met Val Leu Leu Glu Phe Val 210 215 220 ACA GCT GCT GGG ATT ACA CATGGC ATG GAT GAA CTA TAC AAA 714 Thr Ala Ala Gly Ile Thr His Gly Met AspGlu Leu Tyr Lys 225 230 235 TAA 717 238 amino acids amino acid linearprotein 2 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile LeuVal 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val SerGly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys PheIle Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val ThrThr Phe 50 55 60 Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His MetLys Arg 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr ValGln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr ArgAla Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile GluLeu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly HisLys Leu Glu Tyr Asn 130 135 140 Tyr Asn Ser His Asn Val Tyr Ile Met AlaAsp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Val Asn Phe Lys Ile ArgHis Asn Ile Glu Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp His Tyr GlnGln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp AsnHis Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 Lys Asp Pro Asn GluLys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220 Thr Ala Ala GlyIle Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 235 5 amino acids aminoacid <Unknown> linear peptide Modified-site 2 /product= “OTHER” /note=“Xaa = preferably Arg, may be Lys” Modified-site 3 /product= “OTHER”/note= “Xaa = any amino acid” Modified-site 5 /product= “OTHER” /note=“Xaa = hydrophobic amino acid, preferably Val, Leu or Ile” 3 Arg Xaa XaaSer Xaa 1 5 5 amino acids amino acid <Unknown> linear peptideModified-site 2 /product= “OTHER” /note= “Xaa = preferably Arg, may beLys” Modified-site 3 /product= “OTHER” /note= “Xaa = any amino acid”Modified-site 5 /product= “OTHER” /note= “Xaa = hydrophobic amino acid,preferably Val, Leu or Ile” 4 Arg Xaa Xaa Thr Xaa 1 5 13 amino acidsamino acid <Unknown> linear peptide Modified-site 1 /product= “OTHER”/note= “Xaa = Lys or Arg” 5 Xaa Lys Ile Ser Ala Ser Glu Phe Asp Arg ProLeu Arg 1 5 10 5 amino acids amino acid <Unknown> linear peptide 6 AspArg Pro Leu Arg 1 5 8 amino acids amino acid <Unknown> linear peptide 7Xaa Arg Xaa Xaa Ser Xaa Arg Xaa 1 5 9 amino acids amino acid <Unknown>linear peptide 8 Lys Lys Lys Lys Arg Phe Ser Phe Lys 1 5 9 amino acidsamino acid <Unknown> linear peptide 9 Leu Arg Arg Leu Ser Asp Ser AsnPhe 1 5 10 amino acids amino acid <Unknown> linear peptide 10 Lys LysLeu Asn Arg Thr Leu Thr Val Ala 1 5 10 10 amino acids amino acid<Unknown> linear peptide 11 Lys Lys Ala Asn Arg Thr Leu Ser Val Ala 1 510 10 amino acids amino acid <Unknown> linear peptide 12 Met Arg Arg ArgArg Ser Ile Ile Thr Gly 1 5 10 13 amino acids amino acid <Unknown>linear peptide 13 Met Arg Arg Arg Arg Ser Ile Ile Ile Ile Phe Thr Gly 15 10 5 amino acids amino acid <Unknown> linear peptide 14 Arg Arg PheSer Ala 1 5 5 amino acids amino acid <Unknown> linear peptide 15 Lys ArgAsp Ser Met 1 5 9 amino acids amino acid <Unknown> linear peptide 16 MetSer Lys Arg Arg Asp Ser Leu Thr 1 5 18 base pairs nucleic acid singlelinear DNA 17 NNKNNKNNKN NKNNKNNK 18 5 amino acids amino acid <Unknown>linear peptide 18 Arg Arg Leu Ser Ile 1 5 5 amino acids amino acid<Unknown> linear peptide 19 Arg Arg Phe Ser Val 1 5 5 amino acids aminoacid <Unknown> linear peptide 20 Arg Arg Phe Ser Arg 1 5 5 amino acidsamino acid <Unknown> linear peptide 21 Arg Arg Ser Ile Phe 1 5 6 aminoacids amino acid <Unknown> linear peptide 22 Arg Arg Gly Ser Ile Leu 1 56 amino acids amino acid <Unknown> linear peptide 23 Lys Arg Lys Ser GlyIle 1 5 5 amino acids amino acid <Unknown> linear peptide 24 Arg Arg GlySer Val 1 5 8 amino acids amino acid <Unknown> linear peptide 25 Met ArgLys Gly Ser Cys Leu Phe 1 5 8 amino acids amino acid <Unknown> linearpeptide 26 Met Arg Lys Gly Ser Leu Leu Phe 1 5 8 amino acids amino acid<Unknown> linear peptide 27 Met Arg Arg Glu Ser Leu Leu Phe 1 5 8 aminoacids amino acid <Unknown> linear peptide 28 Met Arg Arg Asp Ser Cys LeuPhe 1 5 8 amino acids amino acid <Unknown> linear peptide 29 Met Ser ArgArg Asp Ser Cys Phe 1 5 8 amino acids amino acid <Unknown> linearpeptide 30 Met Ser Lys Arg Arg Asp Ser Leu 1 5 8 amino acids amino acid<Unknown> linear peptide 31 Met Ser Arg Arg Arg Arg Ser Ile 1 5 8 aminoacids amino acid <Unknown> linear peptide 32 Met Arg Arg Arg Arg Ser IleIle 1 5 9 amino acids amino acid <Unknown> linear peptide 33 Met Arg ArgArg Arg Ser Ile Ile Ile 1 5 10 amino acids amino acid <Unknown> linearpeptide 34 Met Arg Arg Arg Arg Ser Ile Ile Ile Phe 1 5 10 11 amino acidsamino acid <Unknown> linear peptide 35 Met Arg Arg Arg Arg Ser Ile IleIle Ile Phe 1 5 10 717 base pairs nucleic acid single linear DNA CDS1..717 /product= “phosphorylation mutant of Aequorea green fluorescentprotein (GPF)” 36 ATG AGT AAA GGA GAA GAA CTT TTC ACT GGA GTT GTC CCAATT CTT GTT 48 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro IleLeu Val 1 5 10 15 GAA TTA GAT GGT GAT GTT AAT GGG AGA AGA TTT TCT GTCAGT GGA GAG 96 Glu Leu Asp Gly Asp Val Asn Gly Arg Arg Phe Ser Val SerGly Glu 20 25 30 GGT GAA GGT GAT GCA ACA TAC GGA AAA CTT ACC CTT AAA TTTATT TGC 144 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe IleCys 35 40 45 ACT ACT GGA AAA CTA CCT GTT CCA TGG CCA ACA CTT GTC ACT ACTTTC 192 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe50 55 60 TCT TAT GGT GTT AGA AGA TTT TCA GCA TAC CCA GAT CAT ATG AAA CGG240 Ser Tyr Gly Val Arg Arg Phe Ser Ala Tyr Pro Asp His Met Lys Arg 6570 75 80 CAT GAC TTT TTC AAG AGT GCC ATG CCC GAA GGT TAT GTA CAG AGA AGA288 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Arg Arg 8590 95 TCT ATA TTT TTC AAA GAT GAC GGG AAC TAC AAG ACA CGT GCT GAA GTC336 Ser Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100105 110 AAG TTT GAA GGT GAT ACC CTT GTT AAT AGA ATC GAG TTA AAA GGT ATT384 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115120 125 GAT TTT AAA AGA AGA GGA TCC ATT CTT GGA CAC AAA TTG GAA TAC AAC432 Asp Phe Lys Arg Arg Gly Ser Ile Leu Gly His Lys Leu Glu Tyr Asn 130135 140 TAT AAC TCA CAC AAT GTA TAC ATC ATG GCA GAC AAA AGA AAG TCT GGA480 Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Arg Lys Ser Gly 145150 155 160 ATC AAA GTT AAC TTC AAA ATT AGA CAC AAC ATT AGA AGA GGA AGCGTT 528 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Arg Arg Gly Ser Val165 170 175 CAA CTA GCA GAC CAT TAT CAA CAA AAT ACT CCA ATT GGC GAT GGCCCT 576 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro180 185 190 GTC CTT TTA CCA GAC AAC AGA AGA CTG TCC ATA CAA TCT GCC CTTTCG 624 Val Leu Leu Pro Asp Asn Arg Arg Leu Ser Ile Gln Ser Ala Leu Ser195 200 205 AAA GAT CCC AAC GAA AAG AGA GAC AGA ATG GTC CTT CTT GAG TTTGTA 672 Lys Asp Pro Asn Glu Lys Arg Asp Arg Met Val Leu Leu Glu Phe Val210 215 220 ACA GCT GCT GGG ATT ACA CAT GGC ATG GAT GAA CTA TAC AAA 714Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 235 TAA717 238 amino acids amino acid linear protein 37 Met Ser Lys Gly Glu GluLeu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly AspVal Asn Gly Arg Arg Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp AlaThr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr Gly Lys LeuPro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 Ser Tyr Gly Val ArgArg Phe Ser Ala Tyr Pro Asp His Met Lys Arg 65 70 75 80 His Asp Phe PheLys Ser Ala Met Pro Glu Gly Tyr Val Gln Arg Arg 85 90 95 Ser Ile Phe PheLys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe GluGly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp PheLys Arg Arg Gly Ser Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 TyrAsn Ser His Asn Val Tyr Ile Met Ala Asp Lys Arg Lys Ser Gly 145 150 155160 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Arg Arg Gly Ser Val 165170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro180 185 190 Val Leu Leu Pro Asp Asn Arg Arg Leu Ser Ile Gln Ser Ala LeuSer 195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp Arg Met Val Leu Leu GluPhe Val 210 215 220 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu TyrLys 225 230 235 129 base pairs nucleic acid single linear DNA CDS13..129 38 GGAGATATAC AT ATG CGG GGT TCT CAT CAT CAT CAT CAT CAT GGT ATG48 Met Arg Gly Ser His His His His His His Gly Met 1 5 10 GCT AGC ATGACT GGT GGA CAG CAA ATG GGT CGG GAT CTG TAC GAC GAT 96 Ala Ser Met ThrGly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp 15 20 25 GAC GAT AAG GATCCC CCC GCT GAA TTC ATG AGT 129 Asp Asp Lys Asp Pro Pro Ala Glu Phe MetSer 30 35 39 amino acids amino acid linear protein 39 Met Arg Gly SerHis His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 Gly Gly GlnGln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 Pro Pro AlaGlu Phe Met Ser 35 61 base pairs nucleic acid single linear DNA 40TACAAATAAT AAGGATCCGA GCTCGAGATC TGCAGCTGGT ACCATGGAAT TCGAAGGTGG 60 A61 153 base pairs nucleic acid single linear DNA 41 GGAGATATACATATGCGGGG TTCTCATCAT CATCATCATC ATGGTATGGC TAGCATGACT 60 GGTGGACAGCAAATGGGTCG GGATCTGTAC GACGATGACG ATAAGGATCC GAGCTCGAG 120 TCTGCAGCTGGTACCATGAG AAGAAGAAGA TCA 153 14 base pairs nucleic acid single linearDNA 42 AAATAAAAGC TTGA 14 10 amino acids amino acid <Unknown> linearpeptide 43 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly 1 5 10 9 amino acidsamino acid <Unknown> linear peptide 44 Met Ser Lys Gly Glu Glu Leu PheThr 1 5 5 amino acids amino acid <Unknown> linear peptide 45 His Lys PheSer Val 1 5 5 amino acids amino acid <Unknown> linear peptideModified-site 3 /product= “OTHER” /note= “Xaa = any amino acid”Modified-site 5 /product= “OTHER” /note= “Xaa = hydrophobic amino acid”46 Arg Arg Xaa Ser Xaa 1 5 5 amino acids amino acid <Unknown> linearpeptide Modified-site 3 /product= “OTHER” /note= “Xaa = any amino acid”Modified-site 5 /product= “OTHER” /note= “Xaa = hydrophobic amino acid”47 Arg Arg Xaa Thr Xaa 1 5 8 amino acids amino acid <Unknown> linearpeptide 48 Met Ser Lys Gly Glu Glu Leu Phe 1 5

What is claimed:
 1. A method for determining whether a sample containsprotein kinase activity comprising; contacting the sample with aphosphate donor and a fluorescent protein substrate for a proteinkinase, the fluorescent protein substrate comprising a fluorescentprotein moiety and a phosphorylation site for a protein kinase, whereinthe fluorescent protein substrate exhibits a different fluorescentproperty in the phosphorylated state than in the un-phosphorylated statewherein the fluorescent protein moiety is a green fluorescent protein(SEQ. ID. NO: 2), and wherein the phosphorylation site is within tenamino acids of the terminus of the fluorescent protein moiety; excitingthe protein substrate; and measuring the amount of a fluorescentproperty of the fluorescent protein substrate that differs in theunphosphorylated state and phosphorylated state, whereby an amount thatis consistent with the presence of the fluorescent protein substrate inits phosphorylated state indicates the presence of protein kinaseactivity.
 2. The method of claim 1, wherein the fluorescent protein isan Aequorea-related fluorescent protein
 3. The method of claim 2,wherein said Aequorea-related fluorescent protein comprises at least onemutation selected from the group consisting of T44A, F64L, V68L, S72A,F99S, Y145F, N1461, M153T, V163A, 1167T, S175G, S205T and N212K.
 4. Themethod of claim 2, wherein said Aequorea-related fluorescent proteincomprises a phosphorylation site within ten amino acids of the terminusof said Aequorea-related fluorescent protein.
 5. The method of claim 2,wherein said Aequorea-related fluorescent protein comprises aphosphorylation site within twenty amino acids of the terminus of saidAequorea-related fluorescent protein.
 6. A method for determiningwhether a cell exhibits protein kinase activity, comprising the stepsof: providing a transfected host cell comprising a recombinant nucleicacid molecule comprising expression control sequences operatively linkedto a nucleic acid sequence coding for the expression of a fluorescentprotein substrate for a protein kinase, the fluorescent proteinsubstrate comprising a fluorescent protein moiety containing aphosphorylation site for a protein kinase, wherein the fluorescentprotein substrate exhibits a different fluorescent property in thephosphorylated state than in the un-phosphorylated state, the cellexpressing the fluorescent protein substrate; wherein the fluorescentprotein moiety is a green fluorescent protein (SEQ. ID. NO: 2), andwherein the phosphorylation site is within ten amino acids of theterminus of the fluorescent protein moiety; exciting the proteinsubstrate in the cell; and measuring the amount of a fluorescentproperty of the fluorescent protein substrate that differs in theun-phosphorylated and phosphorylated states, wherein the presence of thefluorescent property associated with the fluorescent state indicates thepresence of protein kinase activity in the cell.
 7. The method of claim6 wherein the fluorescent protein is an Aequorea-related fluorescentprotein.
 8. The method of claim 7, wherein said Aequorea-relatedfluorescent protein comprises at least one selected from the groupconsisting of T44A, F64L, V68L, S72A, F99S, Y145F, N1461, M153T, V163A,1167T, S175G, S205T and N212K.
 9. The method of claim 7, wherein saidAequorea-related fluorescent protein comprises a phosphorylation sitewithin ten amino acids of the terminus of said Aequorea-relatedfluorescent protein.
 10. The method of claim 7, wherein saidAequorea-related fluorescent protein comprises a phosphorylation sitewithin twenty amino acids of the terminus of said Aequorea-relatedfluorescent protein.
 11. A method for determining whether a compoundalters the activity of a protein kinase, comprising the steps of; 1)contacting a sample comprising a protein kinase activity with thecompound, a phosphate donor for the protein kinase and a fluorescentprotein substrate, wherein the fluorescent protein substrate comprises afluorescent protein moiety and a phosphorylation site for a proteinkinase, and wherein the fluorescent protein substrate exhibits adifferent fluorescent property in the phosphorylated state than in theun-phosphorylated state; wherein the fluorescent protein moiety is agreen fluorescent protein (SEQ. ID. NO: 2), and wherein thephosphorylation site is within ten amino acids of the terminus of thefluorescent protein moiety; 2) exciting the protein substrate; 3)measuring the amount of protein kinase activity in the sample as afunction of the quantity of change or rate of change of a fluorescentproperty of the fluorescent protein substrate that differs in theun-phosphorylated and phosphorylated states; and comparing the amount ofactivity in the sample with a standard activity for the same amount ofsaid protein kinase, whereby a difference between the amount of proteinkinase activity in the sample and the standard activity indicates thatsaid compound has an effect on the activity of the protein kinase. 12.The method of claim 11 wherein the fluorescent protein is anAequorea-related fluorescent protein.
 13. The method of claim 11,wherein said Aequorea-related fluorescent protein comprises at least oneselected from the group consisting of T44A, F64L, V68L, S72A, F99S,Y145F, N1461, M153T, V163A, 1167T, S175G, S205T and N212K.
 14. Themethod of claim 11, wherein said Aequorea-related fluorescent proteincomprises a phosphorylation site within ten amino acids of the terminusof said Aequorea-related fluorescent protein.
 15. The method of claim11, wherein said Aequorea-related fluorescent protein comprises aphosphorylation site within twenty amino acids of the terminus of saidAequorea-related fluorescent protein.